Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Purification and characterization of a lactonase from<Emphasis Type="Italic"> Erwinia cypripedii</Emphasis> 314B that hydrolyzes (<Emphasis Type="Italic">S</Emphasis>)-5-oxo-2-tetrahydrofurancarboxylic acid

A bacterium, strain 314B, able to assimilate (S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l--hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5–7.5 and 35–45 °C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on (R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Psychrotrophic,lactic acid-producing bacteria from anoxic waters in Ace Lake,Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only l(+)lactic acid from d(+)glucose. d(–)ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30°C and were psychrotrophic. Whole cells contained 18:1cis 9 as a major component of their fatty acids. At 20°C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (K_nuc=0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38±8%), and with Carnobacterium mobile (26±2%, 34±2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G+C content of the DNA was 32–34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol or trehalose. The G+C content was 33–34%, and the Type strain is DSM 5972 (=ACAM 313).     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading <Emphasis Type="Italic">Paenibacillus xylanilyticus</Emphasis> KJ-03 and Purification and Characterization of the Recombinant Enzyme

The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.     

<Emphasis Type="Italic">Thermococcus marinus</Emphasis> sp. nov. and<Emphasis Type="Italic"> Thermococcus radiotolerans</Emphasis> sp. nov., two hyperthermophilic archaea from deep-sea hydrothermal vents that resist ionizing radiation

Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23°22N, 44°57W) and the Guaymas Basin (27°01N, 111°24W) at a depth of 2,616 m. Positive enrichments were submitted to -irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1^T was isolated from chimney samples collected from the Mid-Atlantic Ridge after -irradiation at 20 kGy, and strain EJ2^T was isolated from the Guaymas Basin after -irradiation at 30 kGy. Only strain EJ2^T was motile, and both formed regular cocci. These new strains grew between 55 and 95 °C with the optimal temperature being 88 °C. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l^–1. These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1^T, such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2^T was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2^T growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1^T and EJ2^T. The G+C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1^T and EJ2^T, respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA–DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1^T (=DSM 15227^T=JCM 11825^T) and the type strain of T. radiotolerans is strain EJ2^T (=DSM 15228^T=JCM 11826^T).Communicated by J. WiegelThe GenBank accession numbers for the 16S rRNA sequence of Thermococcus marinus strain EJ1^T and Thermococcus radiotolerans EJ2^T are AF479012 and AF479013, respectively.     

Characterization of the cellulase complex from Cellulomonas grown on bagasse pith

Summary The effect of physico-chemical parameters on the cellulolytic activity of Cellulomonas sp. IIbc grown on sugarcane bagasse pith was investigated, and the optimum ranges for enzyme activity were established. The cellulases were more stable when incubated at the optimum growth temperature (32°C) than under optimum activity conditions (45°C for -glucosidases and 50°C for CMC- and FP-cellulases). The -glucosidases were the thermostability-limiting enzymes of the complex. Two types of endoglucanases could be recognized according to their adsorption properties on bagasse: one weakly-bound and one tightly-bound type, the latter constituting approximately 73% of the extracellular endoglucanases at exponential growth phase. Four forms active on filter paper and three active on CMC were obtained by HPLC separation of the extracellular fraction of the culture at stationary phase.Abbreviations CMC carboxymethylcellulose - FP filter paper     

Synthetic esters recognized by glucuronoyl esterase from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.     

Purification,characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from <Emphasis Type="Italic">Curvularia lunata</Emphasis>

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein⁻¹ under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin. This project was supported by the National Natural Science Foundation of China (NSFC; 30572333).     

Production kinetics and stability properties of ϖ(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase is a key enzyme that channels primary metabolites to a tripeptide common to cephalosporin and cephamycin biosynthesis inStreptomyces clavuligerus. Time-course studies indicated that theS. clavuligerus ACV-synthetase was stable during the cephamycin C fermentation: the enzyme was produced early in the growth phase and its activity remained high up to 96 h of growth. The detection of crude ACV-synthetase activity in older cultures was best achieved with an assay medium supplemented with 5 mM phosphoenolpyruvate, at lower ATP concentrations. During storage at 4°C, a progressive decrease in the stability of crude ACV-synthetase was observed with increasing culture age. Although a proteinolytic activity with a pH optimum at 8.2 was detected in crude cell-free extracts, no significant variation was observed in its activity with increasing culture age to account for the instability of ACV-synthetase in vitro. Addition of proteinase inhibitors did not improve the stability of the enzyme. However, a stabilization cocktail containing dithiothreitol. MgCl₂, the three substrate amino acids, and glycerol increased the stability of the enzyme isolated from cultures grown for 30–40 h, which was shortly after the appearance of antibiotics in the culture fluid. This stabilized enzyme retained half of its initial activity after 6 days at 4°C.     

<Emphasis Type="Italic">Halobacillus locisalis</Emphasis> sp. nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea

A Gram-positive, motile, endospore-forming and rod-shaped halophilic bacterial strain MSS-155 (KCTC 3788 and KCCM 41687) was isolated from a marine solar saltern of the Yellow Sea in Korea and was subjected to a polyphasic taxonomic study. This organism grew at temperature of 10.0–42.0°C with an optimum of 35°C. Strain MSS-155 grew optimally in the presence of 10% NaCl and did not grow in the absence of NaCl. The cell wall peptidoglycan type of strain MSS-155 was A4 based on l-Orn-d-Asp. Strain MSS-155 was also characterized chemotaxonomically by having menaquinone-7 (MK-7) as the predominant isoprenoid quinone and anteiso-C_15:0 as the major fatty acid. The DNA G+C content was 44.0 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain MSS-155 falls within the radiation of the cluster comprising Halobacillus species. Levels of 16S rDNA sequence similarity between strain MSS-155 and the type strains of four Halobacillus species were in the range 97.6–98.8%. Strain MSS-155 exhibited levels of DNA-DNA relatedness of 6.2–11.2% to the type strains of Halobacillus species described previously. On the basis of phenotypic properties, phylogeny, and genomic data, strain MSS-155 should be placed in the genus Halobacillus as a member of a novel species, for which we propose the name Halobacillus locisalis sp. nov.Communicated by W.D. Grant     

Transport of maltose by Pseudomonas fluorescens W

The system for uptake of maltose in Pseudomonas fluorescens W was inducible. Using a mutant strain unable to hydrolyze maltose, it was shown that maltose was taken up unaltered against a concentration gradient. Uptake of ¹⁴C maltose was only significantly inhibited by nonradioactive maltose or maltotriose. These were the only sugars that could displace accumulated radioactive maltose in the strain unable to hydrolyze maltose. Uptake exhibited saturation kinetics and was inhibited by energy poisons, indicating that this system was one of active transport. Sulfhydryl-binding reagents reversibly inhibited maltose uptake. No transport ability was lost when cells were subjected to osmotic shock. Using the protein-binding dye 7-diazonium-1,3-naphthalene disulfonate a protein or proteins located in or external to the cell membrane was implicated in maltose transport. The hydrolysis of p-nitrophenyl--D-glucoside (PNPG) was used as an indirect measure of transport ability since penetration of PNPG, not its hydrolysis, was the rate-limiting step.Abbreviations PNPG paranitrophenyl--D-glucoside - NDS 7-diazonium-1,3-naphthalene disulfonic acid - PMB p-hydroxymercuribenzoate - MBS p-chloromercuriphenylsulfonic acid - PCMB p-chloromercuribenzoate - CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Purification and characterization of a complex-bound and a free β-1,4-endoxylanase from the culture fluid of the anaerobic fungus Piromyces sp. strain E2

Two extracellular xylanases were purified to homogeneity from the culture filtrate of the anaerobic fungus Piromyces sp. strain E2 and their properties were studied. The enzymes are present in a High Molecular Mass complex (HMM-complex) and as free protein in nearly equal amounts. Both enzymes are most likely identical as all biochemical characteristics were identical. The molecular masses of the enzymes are 12.5 kDa, as estimated by gel chromatography and electrophoretic mobility. The activities of both enzymes are optimal at pH 6.0 and 50°C and the enzymes are stable up to 72h at 40°C. The enzymes have a pI of 9.1. The K _m and V _max, determined with xylan from oat spelts, were 3 mg · ml^-1 and 2600 IU · mg^-1 protein. The enzymes are active both on soluble and insoluble oat spelt xylan. The purified xylanases are inactive against Avicel, carboxymethylcellulose, p-nitrophenyl--d-glucoside, and p-nitrophenyl--d-xyloside. The products of the pure enzymes are predominantly xylo-oligosaccharides, indicating that the enzymes act as endoxylanases (1,4--d-xylan xylanohydrolases, EC 3.2.1.8).     

Thermus kawarayensis sp. nov., a new member of the genus Thermus, isolated from Japanese hot springs

A long-rod-shaped thermophilic microorganism, strain KW11, was isolated from a hot springs located in the Kawarayu, Gunma, Japan. Cloning and preliminary sequence analysis of 16S rDNA showed that this isolate belongs to the genus Thermus. The cells were 10–20 m long, about 0.8 m in diameter, and produced no pigment in contrast with most of the Thermus species previously reported. KW11 was an aerobic heterotroph and grew at temperatures ranging from 40–73°C, with optimal growth occurring at 68°C. The pH range for growth was from 5.8–8.9, with optimal growth around pH 7. KW11 was sensitive to ampicillin, penicillin G, kanamycin, and streptomycin. The G+C content of DNA was 69 mol%. The main fatty acids were 16:0 (52.9%), iso-15:0 (22.1%), and iso-17:0 (15.6%). The 16S rDNA sequence of KW11 showed 96.0, 95.8, and 95.4% similarity with the sequences of T. aquaticus, T. igniterrae, and T. thermophilus, respectively, and less than 95% with other Thermus species. The physiological differences and phylogenetic evidence indicated that strain KW11 represents T. kawarayensis, a novel species of the genus Thermus. The type strain is isolate KW11^T (JCM12314, DSM16200).     

Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli

Summary The nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl--d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent M _r of approximately 61000. The calculated M _r of the ced1 gene product was 61023.Abbreviations Ap ampicillin - ced1 gene coding for Ced1 - Ced1 cellodextrinase from B. fibrisolvens - CMC carboxymethylcellulose - LB Luria Bertani - ORF open reading frame - pNPC p-nitrophenyl--d-cellobioside - PC phosphate citrate - HCA hydrophobic cluster analysis     

Purification and characterization of an extracellular β-glucosidase from the anaerobic fungus Piromyces sp. strain E2

An extracellular -glucosidase (EC 3.2.2.21) from the anaerobic fungus Piromyces sp. strain E2 was purified. The enzyme is a monomer with a molecular mass of 45 kDa and a pI of 4.15. The enzyme readily hydrolyzes p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, cellobiose, cellotriose, cellotetraose and cellopentaose but is not active towards Avicel, carboxymethylcellulose, xylan, p-nitrophenyl--d-galactoside and p-nitrophenyl--d-xyloside. To cleave p-nitrophenyl--d-glucoside the maximum activity is reached at pH 6.0 and 55°C, and the enzyme is stable up to 72 h at 40°C. Activity is inhibited by d-glucurono--lactone, cellobiose, sodium dodecyl sulfate, Hg²⁺ and Cu²⁺ cations. With p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, and. cellobiose as enzyme substrates, the K _m and V _max balues are 1.5 mM and 25.5 IU·mg^-1, 1.1. mM and 133 IU·mg^-1, and 0.05 mM and 55.6 IU·mg^-1, respectively.     

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn²⁺, Co²⁺ or Mg²⁺, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.